This is a new proposal for studying the molecular mechanisms underlying neurotransmitter secretion. Two complementary approaches will be taken. First, in a "bottom-up" approach, we will reconstitute transmitter secretion mechanisms in two non-neuronal cell types, oocytes and myocytes, in order to determine (1) the components which are necessary and sufficient for efficient calcium-dependent transmitter secretion and (2) the functions served by various presynaptic specific proteins in transmitter secretion. Second, in a "top-down" approach, the status of presynaptic proteins in presynaptic neurons will be manipulated and the effects on synaptic transmission will be examined to determine the functions of these proteins. Two prominent presynaptic proteins, synaptophysin and synapsin I, will be the main focus of the present project, although other presynaptic proteins will be studied when molecular probes become available. In part 1, we will reconstitute an efficient calcium-dependent acetycholine (ACh) secretion mechanism in Xenopus oocytes by a stepwise injection of ACh, purified ACh-containing synaptic vesicles, and mRNA obtained from cholinergic tissue (Torpedo electric lobe) into the oocyte. The requirement of a given presynaptic protein in the secretion process will be examined by studying the effect of co-injection of specific antisense oligonucleotides together with total mRNA of Torpedo electric lobe. In part 2, we will use a voltage- clamped myocyte to detect electrophysiologically both spontaneous and depolarization-evoked ACh secretion from oocytes in order to better characterize the properties of oocyte secretion and to identify the functions of various presynaptic proteins. In part 3, we will reconstitute ACh secretion mechanisms in Xenopus myocytes by a stepwise introduction of ACh, purified synaptic vesicles, and presynaptic proteins. The functions of presynaptic proteins will be determined by examining their effects on the properties of spontaneous and depolarization-evoked ACh secretion, as monitored directly by the myocyte's membrane current induced by its own ACh secretion. In part 4, we will study the roles of synaptophysin and synapsin I in ACh secretion at Xenopus neuromuscular synapses. The status of these proteins in the presynaptic neuron will be altered by over- or reduced-expression of the protein and by protein phosphorylation, and the effect on transmitter secretion will be revealed by examining the changes in the spontaneous and evoked synaptic currents. Taken together, the proposed studies will help to elucidate the functional roles of presynaptic proteins, synaptophysin and synapsin I in particular, in transmitter secretion, and provide insights into the molecular basis of synaptic transmission in the nervous system.